Reading the patterns in living cells —the physics of ca2+ signaling

Ca²⁺ is one of the most important messengers. It transmits signals inside living cells and takes part in intercellular coordination. The dynamics of the Ca²⁺ concentration shows a transition from elemental, stochastic events to global events like waves and oscillations. This transition renders it an ideal tool for studying basic concepts of pattern formation, especially since access to the most important experimental parameters is given. Ca²⁺ dynamics in living cells has been a major topic of biophysical modelling in the last 15 years. Modelling has reached the level of predictive power. The theoretical analysis of waves provided new insight into the mechanisms of Ca²⁺ signaling and led to new concepts of analysis of wave equations with concentration dependent diffusion and novel wave bifurcations. Modelling of oscillations provided understanding especially of complex oscillations and allowed to extract information about the underlying cellular parameters and mechanisms. The investigation of the stochastic aspects of intracellular Ca²⁺ dynamics demonstrated the fundamental role of fluctuations arising from the control of the release channel by Ca²⁺ and IP₃. This review presents an overview of current theoretical research on Ca²⁺ dynamics in living cells driven by the inositol 1,4,5-trisphosphate receptor channel.     

Spatiotemporal dynamics of Ca2+ signaling and its physiological roles

Changes in the intracellular Ca²⁺ concentration regulate numerous cell functions and display diverse spatiotemporal dynamics, which underlie the versatility of Ca²⁺ in cell signaling. In many cell types, an increase in the intracellular Ca²⁺ concentration starts locally, propagates within the cell (Ca²⁺ wave) and makes oscillatory changes (Ca²⁺ oscillation). Studies of the intracellular Ca²⁺ release mechanism from the endoplasmic reticulum (ER) showed that the Ca²⁺ release mechanism has inherent regenerative properties, which is essential for the generation of Ca²⁺ waves and oscillations. Ca²⁺ may shuttle between the ER and mitochondria, and this appears to be important for pacemaking of Ca²⁺ oscillations. Importantly, Ca²⁺ oscillations are an efficient mechanism in regulating cell functions, having effects supra-proportional to the sum of duration of Ca²⁺ increase. Furthermore, Ca²⁺ signaling mechanism studies have led to the development of a method for specific inhibition of Ca²⁺ signaling, which has been used to identify hitherto unrecognized functions of Ca²⁺ signals.     

单个心肌细胞内钙波的微观动力学研究

利用基于近场光学原理构建的全内反射荧光显微镜研究了大鼠单个心肌细胞中的钙信号. 利用这种显微镜的快速成像和高信噪比的特点，观察到单个细胞中复杂的二维钙波斑图. 分析了单个钙信号释放事件在钙波形成、运动过程中的作用. 建立在fire-diffuse-fire模型基础上的模拟显示，由基本钙释放事件组成的钙波可以在心肌细胞中稳定存在. 此研究对进一步认识活体可激发系统的微观动力学行为有指导意义. 关键词： 近场光学 全内反射荧光显微镜 心肌细胞 钙波     

Calcium Ion-Induced Stabilization and Refolding of Agkisacutacin from <Emphasis Type="Italic">Agkistrodon Acutus</Emphasis> Venom Studied by Fluorescent Spectroscopy

Agkisacutacin isolated from the venom of Agkistrodon acutus is a coagulation factor IX / coagulation factor X-binding protein with marked anticoagulant- and platelet-modulating activities. Ca²⁺ ion-induced stabilization and refolding of Agkisacutacin have been studied by following fluorescent measurements. Ca²⁺ ions not only increase the structural stability of agkisacutacin against GdnHCl denaturation, but also induce its refolding. The GdnHCl-induced unfolding of the apo-agkisacutacin and the purified agkisacutacin is a single-step process with no detectable intermediate state. Ca²⁺ ions play an important role in the stabilization of the structure of agkisacutacin. Ca²⁺-stabilized agkisacutacin exhibits higher resistance to GdnHCl denaturation than the apo-agkisacutacin. It is possible to induce refolding of the unfolded apo-agkisacutacin merely by adding 1 mM Ca²⁺ ions without changing the concentration of the denaturant. The kinetic result of Ca²⁺-induced refolding provides evidences for that agkisacutacin consists of at least two refolding phases and the first phase of Ca²⁺-induced refolding should involve the formation of the compact Ca²⁺-binding site regions, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.     

Hydration characteristics of Ca2+ and Mg2+: a density functional theory,polarized continuum model and molecular dynamics investigation

In this work, density functional theory, Møller–Plesset second-order perturbation theory, and ab initio molecular dynamics (AIMD) were used to investigate hydrated characteristics of Mg²⁺ and Ca²⁺ as a function of coordination number in the first hydration shell (CN) and cluster size. It is generally accepted that the CNs of Mg²⁺ and Ca²⁺ are both six. Calculations show that the hydration of Mg²⁺ generally prefers six-coordinated structures, whereas the CN value of Ca²⁺ varies from 6 to 8 as the hydration proceeds. Moreover, the first hydration of Ca²⁺ is found to be more flexible than that of Mg²⁺, as indicated by the results of transition state calculations and AIMD simulations. In addition, the constraint of Mg²⁺ on the first hydration shell is obviously stronger than that of Ca²⁺, while the constraint on the inner hydration shells fades slightly faster for Mg²⁺ than Ca²⁺. It is also found that the charge transfer from central cation to water molecules is affected only by the first hydration shell for Mg²⁺, whereas by the first and second hydration shells for Ca²⁺. Based on hydration characteristics, approximatively saturated ion hydration shells for the hydration of Mg²⁺ and Ca²⁺ were proposed.     

Calcium substituting B-site in relaxor ferroelectrics with perovskite structure probed by chemical ordering

Chemical ordering of Ca²⁺ doped 0.9Pb(Mg_1/3Nb_2/3)O₃-0.1PbTiO₃ ceramics were investigated by dielectric spectra, TEM diffraction and A_1g mode in Raman spectra. It is found degree of relaxor behavior increases first, and then decreases. It conflicts the prediction Ca²⁺ substitutes for A-site ion Pb²⁺ according to crystal chemistry theory. In this letter, a new mechanism that Ca²⁺ substitutes for B-site ions has been proposed, which satisfactorily explained change of chemical ordering. It exhibits strong evidence doped ions with larger ionic radius (Ca²⁺) are quite possibly substitute much smaller ones (Nb⁵⁺ or Ti⁴⁺) in B-site rather than all substitute larger A-site ion in relaxor ferroelectrics.     

Simultaneous registration of Ca2+ transients and volume changes in rat mesangial cells: Evaluation of the effects of protein kinase C down-regulation

The intracellular free Ca²⁺ concentration ([Ca²⁺]_i) could be correlated with the contractile response in rat mesangial cells using an apparatus which measured both biochemical processes simultaneously. Long-term pretreatment of mesangial cells with 12-O-tetradecanoly-phorbol 13-acetate (24 h, 500 nM) increased the (20 nM) angiotensin II-induced mobilization of Ca²⁺ and led to an enhanced and sustained contraction of the cells. The contractile response was delayed by approximately 3.5 s with respect to the intracellular increase in Ca²⁺ concentration. The simultaneous registration of Ca²⁺ transients and cell contractions confirms that [Ca²⁺]_i is the major determinant of the angiotensin II-mediated mesangial cell contraction.Dedicated to Professor Horst H. A. Linde on the occasion of his 60th birthday.     

Intracellular Ca2+ nonlinear wave behaviours in a three dimensional ventricular cell model

Intracellular Ca²⁺ activity regulates a wide range of cellular biochemical processes; in muscle cells, it links membrane excitation to contraction. Ca²⁺ dynamics includes both synchronous oscillations, and nonlinear wave phenomena, both arising from the superposition of spatially localised stochastic events, such as Ca²⁺ sparks. We incorporated individualised cell geometry reconstructed from confocal microscopy with realistic spatial distribution of RyR clusters into the three dimensional ventricular cell model, and reproduced complex spatio-temporal intracellular wave patterns from Ca²⁺ sparks. We also introduced a detailed nuclear Ca²⁺ handing model to simulate prolonged nuclear Ca²⁺ transient, and study the effects of cytosolic-nuclear coupling on intracellular Ca²⁺ dynamics. The model provides a computational platform to study intracellular Ca²⁺ with the ability to interact with experimental measurements of subcellular structures, and can be modified for other cell types.     

Calcifications in human osteoarthritic articular cartilage: ex vivo assessment of calcium compounds using XANES spectroscopy

Calcium (Ca²⁺)‐containing crystals (CCs), including basic Ca²⁺ phosphate (BCP) and Ca²⁺ pyrophosphate dihydrate (CPPD) crystals, are associated with severe forms of osteoarthritis (OA). Growing evidence supports a role for abnormal articular cartilage mineralization in the pathogenesis of OA. However, the role of Ca²⁺ compounds in this mineralization process remains poorly understood. Six patients, who underwent total knee joint replacement for primary OA, have been considered in this study. Cartilage from femoral condyles and tibial plateaus in the medial and lateral compartments was collected as 1 mm‐thick slices cut tangentially to the articular surface. First, CCs presence and biochemical composition were assessed using Fourier transform infrared spectroscopy (FT‐IR). Next, Ca²⁺ compound biochemical form was further assessed using X‐ray absorption spectroscopy (XAS) performed at the Ca²⁺K‐absorption edge. Overall, 12 cartilage samples were assessed. Using FT‐IR, BCP and CPPD crystals were detected in four and three out of 12 samples, respectively. Ca²⁺ compound biochemical forms differed between areas with versus without CCs, when compared using XAS. The complete set of data shows that XANES spectroscopy can be used to accurately characterize sparse CCs in human OA cartilage. It is found that Ca²⁺ compounds differ between calcified and non‐calcified cartilage areas. In calcified areas they appear to be mainly involved in calcifications, namely Ca²⁺ crystals.     

Numerical Simulations of Calcium Ions Spiral Wave in Single Cardiac Myocyte

The calcium ions （Ca^2＋） spark is an elementary Ca^2＋ release event in cardiac myocytes. It is believed to buildup cell-wide Ca^2＋ signals, such as Ca^2＋ transient and Ca^2＋ wave, through a Ca^2＋-induced Ca^2＋ release （CICR） mechanism. Here the excitability of the Ca^2＋ wave in a single cardiac myoeyte is simulated by employing the fire-diffuse-fire model. By modulating the dynamic parameters of Ca^2＋ release and re-uptake channels, we find three Ca^2＋ signaling states in a single cardiac myoeyte： no wave, plane wave, and spiral wave. The period of a spiral wave is variable in the different regimes. This study indicates that the spiral wave or the excitability of the system can be controlled through micro-modulation in a living excitable medium.     

Selective stimulation of catecholamine release from bovine adrenal chromaffin cells by an ionotropic purinergic receptor sensitive to 2-methylthio ATP

Background  

2-Methylthioadenosine 5'-triphosphate (2-MeSATP), formerly regarded as a specific P2Y (metabotropic) purinergic receptor agonist, stimulates Ca²⁺ influx and evokes catecholamine release from adrenal chromaffin cells. These cells express P2Y and P2X (ionotropic) purinoceptors, with the latter providing an important Ca²⁺ influx pathway. Using single cell calcium imaging techniques, we have determined whether 2-MeSATP might be a specific P2X receptor agonist in bovine chromaffin cells and assessed the relative role of P2X and P2Y receptors on catecholamine secretion from these cells.     

Stable cavitation induces increased cytoplasmic calcium in L929 fibroblasts exposed to 1-MHz pulsed ultrasound

An increase in cytoplasmic calcium (Ca²⁺ increase) is a second messenger that is often observed under ultrasound irradiation. We hypothesize that cavitation is a physical mechanism that underlies the increase in Ca²⁺ in these experiments. To control the presence of cavitation, the wave type was controlled in a sonication chamber. One wave type largely contained a traveling wave (wave type A) while the other wave type largely contained a standing wave (wave type B). Fast Fourier transform (FFT) analysis of a sound field produced by the wave types ascertained that stable cavitation was present only under wave type A ultrasound irradiation. Under the two controlled wave types, the increase in Ca²⁺ in L929 fibroblasts was observed with fluorescence imaging. Under wave type A ultrasound irradiation, an increase in Ca²⁺ was observed; however, no increase in Ca²⁺ was observed under wave type B ultrasound irradiation. We conclude that stable cavitation is involved in the increase of Ca²⁺ in cells subjected to pulsed ultrasound.     

Mechano-chemical coupling in spontaneous oscillatory contraction of muscle

Muscle cells take either one of two states, namely contraction (on-state) and relaxation (off-state), under a particular physiological condition (physiological ionic strength, neutral pH and a few mM MgATP). The transition between these two states is regulated by micromolar concentrations of free Ca²⁺. Here we review spontaneous oscillation phenomena named SPOC. The SPOC state is attained in a contractile system of muscle (muscle model without cell membrane) as a third intermediate state. It appears either at an intermediate concentration of free Ca²⁺ (Ca-SPOC) or under the coexistence of MgATP with its hydrolytic products, i.e., MgADP and inorganic phosphate (Pi), where Ca²⁺ is not required (ADP-SPOC). We have constructed a three-dimensional Phase diagram showing three regions corresponding to three states of muscle realized under various concentrations of MgADP, Pi and free Ca²⁺ in the presence of MgATP; the SPOC region was sandwiched between contraction and relaxation regions. We tried to understand the mechano-chemical coupling in SPOC by explaining the mechanical properties of SPOC based on a standard kinetic scheme of actomyosin ATPase; the experimental results could be well simulated, except for the function of Pi, by assuming that a particular kinetic step regulated by Ca²⁺ is also regulated by the feed-back effect of the actomyosin-ADP complex. It is suggested that the SPOC state is attained by cyclic transition among the different chemical states of the actomyosin complex within each half-sarcomere, which occurs spontaneously through the mechanochemical coupling characteristic to the actomyosin complex, i.e., a mechano-enzyme.     

Synchronized Anti-Phase and In-Phase Oscillations of Intracellular Calcium Ions in Two Coupled Hepatocytes System

We study the dynamic behaviour of two intracellufar calcium oscillators that are coupled through gap junctions both to Ca^2＋ and inositol（1,4,5）-trisphosphate （IP3）. It is found that synchronized anti-phase and in-phase oscillations of cytoplasmic cadcium coexist in parameters space. Especially, synchronized anti-phase oscillations only occur near the onset of a Hopf bifurcation point when the velocity of IP3 synthesis is increased. In addition, two kinds of coupling effects, i.e., the diffusions of Ca^2＋ and IP3 among cells on synchronous behaviour, are considered. We fnd that small coupling of Ca^2＋ and large coupling of IP3 facilitate the emergence of synchronized anti-phase oscillations. However, the result is contrary for the synchronized in-phase case. Our findings may provide a qualitative understanding about the mechanism of synchronous behaviour of intercellular calcium signalling.     

Design and Synthesis of a Ruthenium(II) Complex-Based Luminescent Probe for Highly Selective and Sensitive Luminescence Detection of Nitric Oxide

Nitric oxide (NO) is one of the most important intercellular signaling molecules, and plays important roles in various biological systems. In this work, a unique Ru^II complex, tris[(5-(4-methylamino-3-aminobenzylamino)-1,10-phenanthroline)] ruthenium(II) hexafluorophosphate [Ru(MAA-phen)₃][PF₆]₂, has been designed and synthesized as a luminescent probe for the detection of NO in aqueous media. The complex itself is almost non-luminescent, but can specifically react with NO under the aerobic conditions to afford its highly luminescent triazole derivative in aqueous media, [Ru(MTA-phen)₃]²⁺ (MTA-phen: methyl-trazolebenzylamino-1,10-phenanthroline), accompanied by a 302-fold increase in luminescence intensity at 598 nm with a 130 nm Stokes shift. The luminescence response of [Ru(MAA-phen)₃]²⁺ to NO is rapid, highly specific without interferences of other reactive oxygen/nitrogen species, and highly stable against the pH changes in the range of pH 4.5–9.5. These features enable [Ru(MAA-phen)₃]²⁺ to be used as a probe for the highly selective and sensitive luminescence detection of NO in weakly acidic, neutral, and weakly basic media.     

Swelling of K+, Na+ and Ca2+-montmorillonites and hydration of interlayer cations: a molecular dynamics simulation

This paper performs molecular dynamics simulations to investigate the role of the monovalent cations K, Na and the divalent cation Ca on the stability and swelling of montmorillonite. The recently developed CLAYFF force field is used to predict the basal spacing as a function of the water content in the interlayer. The simulations reproduced the swelling pattern of these montmorillonites, suggesting a mechanism of their hydration different (K+ 相似文献   

Downregulation of calcium-dependent NMDA receptor desensitization by sodium-calcium exchangers: a role of membrane cholesterol

Background

The plasma membrane Na⁺/Ca²⁺-exchanger (NCX) has recently been shown to regulate Ca²⁺-dependent N-methyl-d-aspartate receptor (NMDAR) desensitization, suggesting a tight interaction of NCXs and NMDARs in lipid nanoclasters or “rafts”. To evaluate possible role of this interaction we studied effects of Li⁺ on NMDA-elicited whole-cell currents and Ca²⁺ responses of rat cortical neurons in vitro before and after cholesterol extraction by methyl-β-cyclodextrin (MβCD).

Results

Substitution Li⁺ for Na⁺ in the external solution caused a concentration-dependent decrease of steady-state NMDAR currents from 440?±?71 pA to 111?±?29 pA in 140 mM Na⁺ and 140 mM Li⁺, respectively. The Li⁺ inhibition of NMDAR currents disappeared in the absence of Ca²⁺ in the external solution (Ca²⁺-free), suggesting that Li⁺ enhanced Ca²⁺-dependent NMDAR desensitization. Whereas the cholesterol extraction with MβCD induced a decrease of NMDAR currents to 136?±?32 pA in 140 mM Na⁺ and 46?±?15 pA in 140 mM Li⁺, the IC₅₀ values for the Li⁺ inhibition were similar (about 44 mM Li⁺) before and after this procedure. In the Ca²⁺-free Na⁺ solution the steady-state NMDAR currents after the cholesterol extraction were 47?±?6% of control values. Apparently this amplitude decrease was not Ca²⁺-dependent. In the Na⁺ solution containing 1 mM Ca²⁺ the Ca²⁺-dependent NMDAR desensitization was greater when cholesterol was extracted. Obviously, this procedure promoted its development. In agreement, Li⁺ and KB-R7943, an inhibitor of NCX, both considerably reduced NMDA-activated Ca²⁺ responses. The cholesterol extraction itself caused a decrease of NMDA-activated Ca²⁺ responses and, in addition, abolished the effects of Li⁺ and KB-R7943. The cholesterol loading into the plasma membrane caused a recovery of the KB-R7943 effects.

Conclusions

Taken together our data suggest that NCXs downregulate the Ca²⁺-dependent NMDAR desensitization. Most likely, this is determined by a tight functional interaction of NCX and NMDAR molecules because of their co-localization in membrane lipid rafts. The destruction of these rafts is accompanied by an enhancement of NMDAR desensitization and a loss of NCX-selective agent effects on NMDARs.

     

The nestin-expressing and non-expressing neurons in rat basal forebrain display different electrophysiological properties and project to hippocampus

Background

In vertebrates and invertebrates, sensory neurons adapt to variable ambient conditions, such as the duration or repetition of a stimulus, a physiological mechanism considered as a simple form of non-associative learning and neuronal plasticity. Although various signaling pathways, as cAMP, cGMP, and the inositol 1,4,5-triphosphate receptor (InsP₃R) play a role in adaptation, their precise mechanisms of action at the cellular level remain incompletely understood. Recently, in Drosophila, we reported that odor-induced Ca²⁺-response in axon terminals of olfactory receptor neurons (ORNs) is related to odor duration. In particular, a relatively long odor stimulus (such as 5 s) triggers the induction of a second component involving intracellular Ca²⁺-stores.

Results

We used a recently developed in-vivo bioluminescence imaging approach to quantify the odor-induced Ca²⁺-activity in the axon terminals of ORNs. Using either a genetic approach to target specific RNAs, or a pharmacological approach, we show that the second component, relying on the intracellular Ca²⁺-stores, is responsible for the adaptation to repetitive stimuli. In the antennal lobes (a region analogous to the vertebrate olfactory bulb) ORNs make synaptic contacts with second-order neurons, the projection neurons (PNs). These synapses are modulated by GABA, through either GABAergic local interneurons (LNs) and/or some GABAergic PNs. Application of GABAergic receptor antagonists, both GABA_A or GABA_B, abolishes the adaptation, while RNAi targeting the GABAB_R (a metabotropic receptor) within the ORNs, blocks the Ca²⁺-store dependent component, and consequently disrupts the adaptation. These results indicate that GABA exerts a feedback control. Finally, at the behavioral level, using an olfactory test, genetically impairing the GABA_BR or its signaling pathway specifically in the ORNs disrupts olfactory adapted behavior.

Conclusion

Taken together, our results indicate that a relatively long lasting form of adaptation occurs within the axon terminals of the ORNs in the antennal lobes, which depends on intracellular Ca²⁺-stores, attributable to a positive feedback through the GABAergic synapses.     

Recent developments in monitoring calcium and protein interactions in cells using fluorescence lifetime microscopy

Time-resolved fluorescence lifetime microscopy (TRFLM) allows the combination of the sensitivity of fluorescence lifetime to environmental parameters to be monitored in a spatial manner in single living cells, as well as providing more accurate, sensitive, and specific diagnosis of certain clinical diseases and chemical analyses. Here we discuss two applications of TRFLM: (1) the use of nonratiometric probes such as Calcium Crimson, for measuring Ca²⁺; and (2) quantification of protein interaction in living cells using green and blue fluorescent protein (GFP and BFP, respectively) expressing constructs in combination with fluorescence resonance energy transfer microscopy (FRET). With respect to measuring Ca²⁺ in biological samples, we demonstrate thatintensity-based measurements of Ca²⁺ with single-wavelength Ca²⁺ probes such as Calcium Crimson may falsely report the actual Ca²⁺ concentration. This is due to effects of hydrophobicity of the local environment on the emission of Calcium Crimson as well as interaction of Calcium Crimson with proteins, both of which are overcome by the use of TRFLM. The recent availability of BFP (P4-3) and GFP (S65T) (which can serve as donor and acceptor, respectively) DNA sequences which can be attached to the carboxy-or amino-terminal DNA sequence of specific proteins allows the dual expression and interaction of proteins conjugated to BFP and GFP to be monitored in individual cells using FRET. Both of these applications of TRFLM are expected to enhance substantially the information available regarding both the normal and the abnormal physiology of cells and tissues.